Field of the Invention
The invention relates to methods useful for manufacture of RNA transcripts, e.g., mRNA.
Description of the Related Art
The manufacturing process used to produce clinical grade chemically modified mRNA therapeutics must be able to generate mRNA of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). The RNA must be as homogeneous as possible; this includes obtaining a uniform cap structure/5′ terminus, correct sequence, correct poly A tail length and minimizing the formation of product related impurities.
To generate poly A tail containing mRNAs, several approaches have been taken utilizing in vitro transcription. The first is transcription using a “tailless” DNA template. A 3′ Poly A tail is added, post-transcriptionally using a Poly A polymerase. Tails greater than 100 bases are typically generated. The downfall of this approach is the difficulty of controlling tail length and the evolution of tail length distribution in the RNA.
The use of PCR to generate DNA templates containing a Poly A:T tract is another approach. The poly A:T tracts are introduced via PCR primers, introducing another step for creation of impurities.